The plasma membrane is a key interface between the cell interior and the outside environment. The membrane bilayer is a mixture of lipids with embedded membrane proteins; recent evidence indicates there are lateral heterogeneities in the cell membrane, microdomains termed lipid rafts that are enriched in sphingolipids and cholesterol. The rafts recruit a distinct subset of membrane proteins, including GPI-anchored proteins and many lipid-modified signaling proteins such as src family kinases and G proteins. The rafts are small, dynamic structures, but they can be organized into 50-100 nm flask-shaped plasma membrane invaginations termed caveolae by caveolin family proteins. The long-range objectives of these studies is to elucidate the molecular basis for the biogenesis of rafts and caveolae and to understand their role in cellular function. In particular, the Specific Aims of this proposed project are to (1) distinguish between targeting of proteins to lipid rafts and to caveolae; (2) determine how crosslinking by ligand or antibody affects movement between bulk plasma membrane, lipid rafts, and caveolae; and (3) characterize the structural features of caveolin- 1 and caveolin-2 that are required for formation of caveolae. These studies will be accomplished through expression of various raft proteins, caveolin isoforms, and a variety of caveolin mutants in cell lines, with some of these constructs tagged with green fluorescent protein (GFP) for fluorescence studies. The cells will be studied with biochemical techniques along with confocal fluorescence microscopy, immunogold electron microscopy, freeze-fracture, deep-etch electron microscopy to visualize the detailed structure of caveolae, and fluorescence resonance energy transfer (FRET) to provide a high-resolution dynamic picture of the protein-protein interaction and movements within and between rafts and caveolae. Changes induced during cell signaling through GPI-anchored proteins will be investigated. Overall, the importance of this research to health and disease is highlighted by the diverse role of rafts and caveolae in an array of pathophysiological processes: caveolae have been shown to exert growth regulatory effects (genetic mapping data suggest a possible role of caveolin- 1 as a tumor suppressor gene), caveolin-1 binds cholesterol and has been implicated in cholesterol movement within the cell, and multiple signaling pathways have been shown to involve rafts, including signal transduction through the T and B cell receptors.